PROJECT SUMMARY/ABSTRACT Many studies have shown a correlation between the sphingolipid signaling pathway and cancer pathogenesis as well as drug-resistance. The central molecule in sphingolipid pathway, ceramide, is involved in the induction of apoptosis, senescence, and growth arrest in many human cancers including breast cancer. Recent studies have shown that certain ceramide analogs possess the capability of preferentially killing resistant cancer cells and/or inhibiting glucosylceramide synthase (GCS) activity, which is highly correlated to cancer multidrug-resistance. The main hypothesis of this project is that ceramide analogs can be developed as new therapeutic agents for the treatment of chemo-resistant and aggressive metastatic breast cancers influencing clinical cancer treatment concepts. Our Secondary hypothesis is that self-fluorescent ceramide analogs can be used to affirm known ceramide mechanisms of action, determine potential new mechanisms of action, and discover novel targets and interactions. Four specific aims are proposed for the Project: Specific Aim 1 - Development of highly potent selective agents for killing resistant cancer cells as candidates for clinical treatment of breast cancer multidrug-resistance and metastasis; Specific Aim 2 - Determination of the mechanistic factors leading to the apoptosis of resistant and metastatic cells, and investigating the relationship between preferential killing of resistant cancer cells and glucosylceramide synthase (GCS) and ceramidase inhibition by ceramide analogs; Specific Aim 3 - Using a novel experimental tool, self-fluorescent small molecules, for investigating the distribution and action of ceramides in cancer cells; and Specific Aim 4 - Determination of the in vivo effects of the most potent target ceramide analog(s). To achieve the Specific Aim 1, 40-45 self-fluorescent ceramide analogs will be synthesized with extended aromatic conjugated systems. To achieve the Specific Aim 2, general anticancer activity screenings will be performed to identify the effective and selective ceramide analogs. A series of mechanistic studies on the potent ceramide analogs will be performed to examine whether the analogs induce apoptosis, and if yes, through which pathway(s). A glucosylceramide level assay, GCS and P-gp level assays, resistance-reversal assays, and ceramidase inhibition assays will determine the effects of the target analogs on the activity and expression of GCS and P-gp and ceramidase. To achieve the Specific Aim 3, through fluorescence microscopy, distribution and actions of the target fluorescent ceramide analogs will be tracked in treated cells, validating the mechanisms of action. Finally, most potent analogs will be used in animal studies to achieve the Specific Aim 4. The long-term objective of this project is to improve the survival rate and quality of life of breast cancer patients by targeting the ceramide signaling pathway. Undergraduate research students, including participants in research training programs, will be involved in all aspects of the Project.